A novel bispecific antibody drug conjugate targeting HER2 and HER3 with potent therapeutic efficacy against breast cancer.

Antibody drug conjugate (ADC) therapy has become one of the most promising approaches in cancer immunotherapy. Bispecific targeting could enhance the efficacy and safety of ADC by improving its specificity, affinity and internalization. In this study we constructed a HER2/HER3-targeting bispecific ADC (BsADC) and characterized its physiochemical properties, target specificity and internalization in vitro, and assessed its anti-tumor activities in breast cancer cell lines and in animal models. The HER2/HER3-targeting BsADC had a drug to antibody ratio (DAR) of 2.89, displayed a high selectivity against the target JIMT-1 breast cancer cells in vitro, as well as a slightly higher level of internalization than HER2- or HER3-monospecific ADCs. More importantly, the bispecific ADC potently inhibited the viability of MCF7, JIMT-1, BT474, BxPC-3 and SKOV-3 cancer cells in vitro. In JIMT-1 breast cancer xenograft mice, a single injection of bispecific ADC (3 mg/kg, i.v.) significantly inhibited the tumor growth with an efficacy comparable to that caused by combined injection of HER2 and HER3-monospecific ADCs (3 mg/kg for each). Our study demonstrates that the bispecific ADC concept can be applied to development of more potent new cancer therapeutics than the monospecific ADCs.

T cell-redirecting antibody for treatment of solid tumors via targeting mesothelin.

T cell engaging bispecific antibodies (TCBs) have recently become significant in cancer treatment. In this study we developed MSLN490, a novel TCB designed to target mesothelin (MSLN), a glycosylphosphatidylinositol (GPI)-linked glycoprotein highly expressed in various cancers, and evaluated its efficacy against solid tumors. CDR walking and phage display techniques were used to improve affinity of the parental antibody M912, resulting in a pool of antibodies with different affinities to MSLN. From this pool, various bispecific antibodies (BsAbs) were assembled. Notably, MSLN490 with its IgG-[L]-scFv structure displayed remarkable anti-tumor activity against MSLN-expressing tumors (EC50: 0.16 pM in HT-29-hMSLN cells). Furthermore, MSLN490 remained effective even in the presence of non-membrane-anchored MSLN (soluble MSLN). Moreover, the anti-tumor activity of MSLN490 was enhanced when combined with either Atezolizumab or TAA × CD28 BsAbs. Notably, a synergistic effect was observed between MSLN490 and paclitaxel, as paclitaxel disrupted the immunosuppressive microenvironment within solid tumors, enhancing immune cells infiltration and improved anti-tumor efficacy. Overall, MSLN490 exhibits robust anti-tumor activity, resilience to soluble MSLN interference, and enhanced anti-tumor effects when combined with other therapies, offering a promising future for the treatment of a variety of solid tumors. This study provides a strong foundation for further exploration of MSLN490's clinical potential.

Genome-Wide Association Study Reveals Polygenic Architecture for Limber Pine Quantitative Disease Resistance to White Pine Blister Rust.

Development of durable resistance effective against a broad range of pathotypes is crucial for restoration of pathogen-damaged ecosystems. This study dissected the complex genetic architecture for limber pine quantitative disease resistance (QDR) to Cronartium ribicola using a genome-wide association study. Eighteen-month-old seedlings were inoculated for resistance screening under controlled conditions. Disease development was quantitatively assessed for QDR-related traits over 4 years postinoculation. To reveal the genomic architecture contributing to QDR-related traits, a set of genes related to disease resistance with genome-wide distribution was selected for targeted sequencing for genotyping of single-nucleotide polymorphisms (SNPs). The genome-wide association study revealed a set of SNPs significantly associated with quantitative traits for limber pine QDR to white pine blister rust, including number of needle spots and stem cankers, as well as survival 4 years postinoculation. The peaks of marker-trait associations displayed a polygenic pattern, with genomic regions as potential resistant quantitative trait loci, distributed over 10 of the 12 linkage groups (LGs) of Pinus. None of them was linked to the Cr4-controlled major gene resistance previously mapped on LG08. Both normal canker and bole infection were mapped on LG05, and the associated SNPs explained their phenotypic variance up to 52%, tagging a major resistant quantitative trait locus. Candidate genes containing phenotypically associated SNPs encoded putative nucleotide-binding site leucine-rich repeat proteins, leucine-rich repeat-receptor-like kinase, cytochrome P450 superfamily protein, heat shock cognate protein 70, glutamate receptor, RNA-binding family protein, and unknown protein. The confirmation of resistant quantitative trait loci broadens the genetic pool of limber pine resistance germplasm for resistance breeding.

Long-term passaging of pseudo-typed SARS-CoV-2 reveals the breadth of monoclonal and bispecific antibody cocktails.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses challenges to the effectiveness of neutralizing antibodies. Rational design of antibody cocktails is a realizable approach addressing viral immune evasion. However, evaluating the breadth of antibody cocktails is essential for understanding the development potential. Here, based on a replication competent vesicular stomatitis virus model that incorporates the spike of SARS-CoV-2 (VSV-SARS-CoV-2), we evaluated the breadth of a number of antibody cocktails consisting of monoclonal antibodies and bispecific antibodies by long-term passaging the virus in the presence of the cocktails. Results from over two-month passaging of the virus showed that 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 from these cocktails were highly resistant to random mutation, and there was no breakthrough after 30 rounds of passaging. As a control, antibody REGN10933 was broken through in the third passage. Next generation sequencing was performed and several critical mutations related to viral evasion were identified. These mutations caused a decrease in neutralization efficiency, but the reduced replication rate and ACE2 susceptibility of the mutant virus suggested that they might not have the potential to become epidemic strains. The 9E12 + 10D4 + 2G1 and 7B9-9D11 + 2G1 cocktails that picked from the VSV-SARS-CoV-2 system efficiently neutralized all current variants of concern and variants of interest including the most recent variants Delta and Omicron, as well as SARS-CoV-1. Our results highlight the feasibility of using the VSV-SARS-CoV-2 system to develop SARS-CoV-2 antibody cocktails and provide a reference for the clinical selection of therapeutic strategies to address the mutational escape of SARS-CoV-2.

Comparative Association Mapping Reveals Conservation of Major Gene Resistance to White Pine Blister Rust in Southwestern White Pine (Pinus strobiformis) and Limber Pine (P. flexilis).

All native North American white pines are highly susceptible to white pine blister rust (WPBR) caused by Cronartium ribicola. Understanding genomic diversity and molecular mechanisms underlying genetic resistance to WPBR remains one of the great challenges in improvement of white pines. To compare major gene resistance (MGR) present in two species, southwestern white pine (Pinus strobiformis) Cr3 and limber pine (P. flexilis) Cr4, we performed association analyses of Cr3-controlled resistant traits using single nucleotide polymorphism (SNP) assays designed with Cr4-linked polymorphic genes. We found that ∼70% of P. flexilis SNPs were transferable to P. strobiformis. Furthermore, several Cr4-linked SNPs were significantly associated with the Cr3-controlled traits in P. strobiformis families. The most significantly associated SNP (M326511_1126R) almost colocalized with Cr4 on the Pinus consensus linkage group 8, suggesting that Cr3 and Cr4 might be the same R locus, or have localizations very close to each other in the syntenic region of the P. strobiformis and P. flexilis genomes. M326511_1126R was identified as a nonsynonymous SNP, causing amino acid change (Val376Ile) in a putative pectin acetylesterase, with coding sequences identical between the two species. Moreover, top Cr3-associated SNPs were further developed as TaqMan genotyping assays, suggesting their usefulness as marker-assisted selection (MAS) tools to distinguish genotypes between quantitative resistance and MGR. This work demonstrates the successful transferability of SNP markers between two closely related white pine species in the hybrid zone, and the possibility for deployment of MAS tools to facilitate long-term WPBR management in P. strobiformis breeding and conservation.

A new pair of cytotoxic enantiomeric isoprenylated chromone derivatives from Pestalotiopsis sp.

A new pair of enantiomeric isoprenylated chromone derivatives, (±)-pestaloficiol X [(±)-1], along with a known compound pestaloficiol J (2), were isolated from the plant endophytic fungus Pestalotiopsis sp. The racemic mixture 1 was separated through chiral HPLC. The structures of new compounds (±)-1 were elucidated on the basis of extensive spectroscopic data and their absolute configurations were further configured through computational analysis of their electronic circular dichroism (ECD) spectra. Compound (+)-1 showed significant inhibitory potency against HL-60 and HEP-3B cell lines, with IC50 values of 1.35 ± 0.15 and 3.70 ± 0.33 µM, respectively, while compound (-)-1 showed significant inhibitory potency against HL-60 and HEP-3B cell lines, with IC50 values of 2.39 ± 0.26 and 2.99 ± 0.35 µM, respectively.

Fine dissection of limber pine resistance to Cronartium ribicola using targeted sequencing of the NLR family.

BACKGROUND: Proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains (NLR) make up one of most important resistance (R) families for plants to resist attacks from various pathogens and pests. The available transcriptomes of limber pine (Pinus flexilis) allow us to characterize NLR genes and related resistance gene analogs (RGAs) in host resistance against Cronartium ribicola, the causal fungal pathogen of white pine blister rust (WPBR) on five-needle pines throughout the world. We previously mapped a limber pine major gene locus (Cr4) that confers complete resistance to C. ribicola on the Pinus consensus linkage group 8 (LG-8). However, genetic distribution of NLR genes as well as their divergence between resistant and susceptible alleles are still unknown. RESULTS: To identify NLR genes at the Cr4 locus, the present study re-sequenced a total of 480 RGAs using targeted sequencing in a Cr4-segregated seed family. Following a call of single nucleotide polymorphisms (SNPs) and genetic mapping, a total of 541 SNPs from 155 genes were mapped across 12 LGs. Three putative NLR genes were newly mapped in the Cr4 region, including one that co-segregated with Cr4. The tight linkage of NLRs with Cr4-controlled phenotypes was further confirmed by bulked segregation analysis (BSA) using extreme-phenotype genome-wide association study (XP-GWAS) for significance test. Local tandem duplication in the Cr4 region was further supported by syntenic analysis using the sugar pine genome sequence. Significant gene divergences have been observed in the NLR family, revealing that diversifying selection pressures are relatively higher in local duplicated genes. Most genes showed similar expression patterns at low levels, but some were affected by genetic background related to disease resistance. Evidence from fine genetic dissection, evolutionary analysis, and expression profiling suggests that two NLR genes are the most promising candidates for Cr4 against WPBR. CONCLUSION: This study provides fundamental insights into genetic architecture of the Cr4 locus as well as a set of NLR variants for marker-assisted selection in limber pine breeding. Novel NLR genes were identified at the Cr4 locus and the Cr4 candidates will aid deployment of this R gene in combination with other major/minor genes in the limber pine breeding program.

In-vitro anti-fungal assay and association analysis reveal a role for the Pinus monticola PR10 gene (PmPR10-3.1) in quantitative disease resistance to white pine blister rust.

Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of conifer trees. Here, we identified multiple novel members in the western white pine (Pinus monticola) PmPR10 family by bioinformatic mining available transcriptomic data. Phylogenetic analysis of protein sequences revealed four PR10 and two PR10-like clusters with a high synteny across different species of five-needle pines. Of 10 PmPR10 genes, PmPR10-3.1 was selected and expressed in Escherichia coli. The purified recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens Cronartium ribicola, Phoma exigua, and Phoma argillacea by in-vitro anti-fungal analysis. Genetic variation analysis detected a total of 21 single nucleotide polymorphisms (SNPs) within PmPR10-3.1 in a collection of P. monticola seed families. A nonsynonymous SNP (t178g) showed significant association with relative levels of quantitative disease resistance (QDR), explaining about 8.7% of phenotypic variation as the peak value across all SNPs. Our results provide valuable insight into the genetic architecture underlying P. monticola QDR and imply that PmPR10-3.1 may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.

Limber pine (Pinus flexilis James) genetic map constructed by exome-seq provides insight into the evolution of disease resistance and a genomic resource for genomics-based breeding.

Limber pine (Pinus flexilis) is a keystone species of high-elevation forest ecosystems of western North America, but some parts of the geographic range have high infection and mortality from the non-native white pine blister rust caused byCronartium ribicola. Genetic maps can provide essential knowledge for understanding genetic disease resistance as well as local adaptation to changing climates. Exome-seq was performed to construct high-density genetic maps in two seed families. Composite maps positioned 9612 unigenes across 12 linkage groups (LGs). Syntenic analysis of genome structure revealed that the majority of orthologs were positional orthologous genes (POGs) with localization on homologousLGs among conifer species. Gene ontology (GO) enrichment analysis showed relatively fewer constraints forPOGs with putative roles in adaptation to environments and relatively more conservation forPOGs with roles in basic cell function and maintenance. The mapped genes included 639 nucleotide-binding site leucine-rich repeat genes (NBS-LRRs), 290 receptor-like protein kinase genes (RLKs), and 1014 genes with potential roles in the defense response and induced systemic resistance to attack by pathogens. Orthologous loci for resistance to rust pathogens were identified and were co-positioned with multiple members of theR gene family, revealing the evolutionary pressure acting upon them. This high-density genetic map provides a genomic resource and practical tool for breeding and genetic conservation programs, with applications in genome-wide association studies (GWASs), the characterization of functional genes underlying complex traits, and the sequencing and assembly of the full-length genomes of limber pine and relatedPinus species.

Genetic diversity of strawberry mild yellow edge virus from eastern Canada.

Strawberry mild yellow edge virus (SMYEV) is a member of the genus Potexvirus, family Alphaflexiviridae. It is one of the most common pathogenic viruses infecting cultivated strawberries worldwide. In this study, we investigated the genetic diversity of SMYEV in strawberry fields that were severely affected by strawberry decline disease in the eastern Canadian provinces of New Brunswick, Nova Scotia, Prince Edward Island and Quebec. A total of 134 SMYEV coat protein (CP) gene sequences, representing 85 nucleic acid haplotypes, were identified in 56 field samples. A highly divergent SMYEV population was found in all four provinces, but there was little genetic differentiation among the populations, and moreover, the Canadian SMYEV isolates formed a unique dissimilar, genetically divergent population group when compared to those reported in other countries. Phylogenetic analysis revealed three new SMYEV subclades that consisted mainly of Canadian variants and were composed of 76 sequence haplotypes (76/85, 88%). Mixed infections by different SMYEV variants were observed in 38 samples (38/56, 68%). Evolutionary analysis suggested that the SMYEV strains in eastern Canada possibly originated outside of Canada but adapted to conditions in the region through genetic mutations.

Emerging role for kynurenines in metabolic pathologies.

PURPOSE OF REVIEW: So far, the tryptophan catabolites generated in the kynurenine pathway have been mainly studied in relation to oncologic and mental health disorders. The current review provides an update on the emerging biomedical interest for kynurenine pathway activity in the field of energy homeostasis and metabolic diseases. RECENT FINDINGS: Kynurenine pathway enzymes are expressed in tissues relevant for energy homeostasis such as fat, skeletal muscle, liver and endocrine pancreas, blood vessel and heart, and are regulated by nutritional and inflammatory signals. Kynurenine pathway metabolites have been proposed as biomarkers for initiation and progression of atherosclerosis and diabetes. Exercise training activation of kynurenine pathway in skeletal muscles increases lipid metabolism and thermogenesis, and it limits weight gain, inflammation, insulin resistance, and glucose intolerance in rodents fed a high-fat diet. Manipulation of kynurenine pathway metabolism through administration of enzyme inhibitors or kynurenine pathway metabolites can serve as novel therapeutic strategy for atherosclerosis, obesity, glucose intolerance, or impaired insulin secretion. SUMMARY: Although we are far from a complete understanding of the role of kynurenine pathway in the modulation of energy homeostasis, targeting kynurenine pathway harbors high potential to expand the range of therapies to prevent and treat metabolic diseases.

Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence.

BACKGROUND: Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS: Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS: In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION: Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

Characterization of the western white pine TIR-NBS-LRR (PmTNL2) gene by transcript profiling and promoter analysis.

Proteins with nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) have been reported to play important roles in plant disease resistance, growth, and development. However, no comprehensive analysis of this protein family has been performed in conifers. Here we report that the Pinus monticola PmTNL2 gene is a member of the NBS-LRR superfamily. Quantitative reverse transcription-PCR (qRT-PCR) analysis revealed that the PmTNL2 transcript was expressed in a tissue-specific pattern with extensive regulation by various environmental stimuli in western white pine seedlings, suggesting its wide involvement in stress defense and diverse developmental processes. In silico analysis of the PmTNL2 promoter region revealed multiple cis-regulatory elements characterized with potential functions for development-, light-, and stress-regulated transcript expression. Expression patterns were largely confirmed by PmTNL2 promoter-directed reporter gene expression using stable transgenic Arabidopsis plants. Notably, the PmTNL2 promoter activity was highly expressed in shoot apical and floral meristems and was induced strongly with vascular specificity by pathogen infection. Our data has provided a fundamental insight into both expression regulation and putative functions of the PmTNL2 gene in the context of plant growth and development, as well as in responses to environmental stressors. Promoter application as a potential tool for tree improvement was further discussed.

Xylomolones A-D from the Thai Mangrove Xylocarpus moluccensis: Assignment of Absolute Stereostructures and Unveiling a Convergent Strategy for Limonoid Biosynthesis.

Two new 9,10- seco limonoids with a central 3,4-dihydro-2 H-pyran motif, named xylomolones A and B (1 and 2, respectively), possessing identical absolute configurations but reversed alignments of ring A, compared to the tricyclic core of rings B-D, were obtained from seeds of the Thai mangrove Xylocarpus moluccensis, together with a highly modified 1,2- seco limonoid, named xylomolone C (3), containing a novel 3-oxabicyclo[3.2.1]octane-2,7-dione motif, and a new C11-terpenic acid methyl ester (4). The relative and absolute configurations of 1-4 were evidenced by extensive nuclear magnetic resonance (NMR) investigations combined with density functional theory and time-dependent density functional theory calculations of electronic circular dichroism, specific optical rotation, and 13C NMR data. The co-isolation of 1-4 allowed us to propose a novel convergent strategy for limonoid biosynthesis for the first time. This study demonstrates that mangroves of the genus Xylocarpus harbor new limonoid biosynthetic routes.

Identification and Functional Characterization of an Effector Secreted by Cronartium ribicola.

Cri-9402 was identified as a protein effector from Cronartium ribicola, based on the effect of its expression on growth of Pseudomonas syringae Psm ES4326 introduced into transiently transformed tobacco leaves and stably transformed Arabidopsis seedlings. In tobacco leaves transiently expressing its coding sequence, growth of P. syringae Psm ES4326 was inhibited. Expression of pathogenesis-related (PR) protein 2 (PR2), PR4a, endochitinase B, hypersensitive-related 201 (HSR201), HSR203J, and proteinase inhibitor 1 was upregulated but expression of PR1, coronatine insensitive 1, and abscisic acid 1 was significantly suppressed. In transformed Arabidopsis seedlings, the effector stimulated growth of P. syringae Psm ES4326; significantly suppressed expression of PR1, PR2, nonexpresser of pathogenesis-related genes 1 (NPR1), NPR3, NPR4, phytoalexin deficient 4, and salicylic acid induction deficient 2; and enhanced expression of plant defensin 1.2 (PDF1.2). The above results showed that the majority of responses to this effector in tobacco leaves were converse to those in transformed Arabidopsis. We could conclude that Cri-9402 promoted disease resistance in tobacco leaves and disease susceptibility in Arabidopsis seedlings. Its transcript was mainly expressed in aeciospores of C. ribicola and was probably involved in production or germination of aeciospores, and it was an effector potentially functioning in white pine-blister rust interactions.

Alkaloids with Immunosuppressive Activity from the Bark of Pausinystalia yohimbe.

Ten new alkaloids (1-10), including two pairs of enantiomeric mixtures (5a,b and 6a,b), and 15 known analogues (11-25) were obtained from the bark of Pausinystalia yohimbe. The structures of 1-25 were established by spectroscopic methods, and the absolute configurations of compounds 1-10 were resolved by X-ray diffraction and ECD data analyses. The in vitro immunosuppressive activities of selected isolates were tested. Compounds 11 and 16 exhibited moderate inhibition with IC50 values of 16.8 and 27.6 µM against ConA-induced T lymphocyte proliferation and 13.5 and 40.5 µM against LPS-induced B lymphocyte proliferation, respectively.

Triangular Silver Nanoplate-Coated ZnO Nanoflowers Photocatalysis of Norfloxacin Under Visible Light.

T-Ag/ZnO nanoflowers were successfully fabricated via two steps methods on zinc foil. The chemical composition of norfloxacin was investigated by FTIR spectroscopy. The morphology, composition, and structural and optical properties of the as-synthesized materials were characterized. The results show that triangular silver nanoplates exhibit unique surface plasmon resonance (SPR) absorption spectra, and the absorption spectrum range of ZnO nanoflowers are effectively expanded by coating triangular silver nanoplates. The photocatalytic degradation of norfloxacin activity can be obviously improved because of a synergetic effect and unique SPR of triangular silver nanoplates in the T-Ag/ZnO nanoflowers under visible light. In addition, the possible mechanism for T-Ag/ZnO nanoflowers for the photodegradation of norfloxacin are discussed. The stability of T-Ag/ZnO nanoflowers are also studied.

Profiling methyl jasmonate-responsive transcriptome for understanding induced systemic resistance in whitebark pine (Pinus albicaulis).

KEY MESSAGE: RNA-seq analysis on whitebark pine needles demonstrated that methyl jasmonate (MeJA)-triggered transcriptome re-programming substantially overlapped with defense responses against insects and fungal pathogens in Pinus species, increasing current knowledge regarding induced systemic resistance (ISR) to pathogens and pests in whitebark pine. Many whitebark pine populations are in steep decline due to high susceptibility to mountain pine beetle and the non-native white pine blister rust (WPBR). Resistance, including induced systemic resistance (ISR), is not well characterized in whitebark pine, narrowing the current options for increasing the success of restoration and breeding programs. Exogenous jasmonates are known to trigger ISR by activating the plant's immune system through regulation of gene expression to produce chemical defense compounds. This study reports profiles of whitebark pine needle transcriptomes, following methyl jasmonate (MeJA) treatment using RNA-seq. A MeJA-responsive transcriptome was de novo assembled and transcriptome profiling identified a set of differentially expressed genes (DEGs), revealing 1422 up- and 999 down-regulated transcripts with at least twofold change (FDR corrected p < 0.05) in needle tissues in response to MeJA application. GO analysis revealed that these DEGs have putative functions in plant defense signalling, transcription regulation, biosyntheses of secondary metabolites, and other biological processes. Lineage-specific expression of defense-related genes was characterized through comparison with MeJA signalling in model plants. In particular, MeJA-triggered transcriptome re-programming substantially overlapped with defense responses against WPBR and insects in related Pinus species, suggesting that MeJA may be used to improve whitebark pine resistance to pathogens/pests. Our study provides new insights into molecular mechanisms and metabolic pathways involved in whitebark pine ISR. DEGs identified in this study can be used as candidates to facilitate identification of genomic variation contributing to host resistance and aid in breeding selection of elite genotypes with better adaptive fitness to environmental stressors in this endangered tree species.

Illustrating the Mass-Transport Effect on Enzyme Cascade Reaction Kinetics by Use of a Rotating Ring-Disk Electrode.

Electrochemical biosensors based on enzymatic reaction have been applied to a wide range of fields. As the trend continues to grow, these biosensors are approaching the limit imposed by physics and chemistry. To further improve the performance of biosensors, the interplay of mass transport and enzymatic reaction kinetics, especially in enzyme cascade systems, should be considered in the design of biosensors. Herein, we propose a simple approach to studying the influence of mass transport and enzyme molecule motion on the kinetics of enzyme cascade reactions. ß-Galactosidase (ß-Gal) and glucose oxidase (GOx) of the enzyme cascade reaction are precisely immobilized onto the disk and ring electrodes, respectively, of a rotating ring-disk electrode (RRDE) via covalent attachment. At a low rotating speed (<600 rpm), convective transport promotes the enzyme cascade reaction. When the rotating speed is higher than 600 rpm, the cascade reaction becomes kinetically controlled. Further increase of the rotating speed results in a slow decline in reaction rate, possibly due to the production inhibition effect. In addition, the effect of conformation change of the enzyme at higher centrifugal forces on enzyme activity should be considered. This study would shine light on the effect of convective force on regulation of kinetics of enzyme cascade reaction, offering an ideal platform for studying other enzyme cascade reactions and providing fundamentals to design high-performance biosensors, biofuel cells, and bioelectronics.

Saturated genic SNP mapping identified functional candidates and selection tools for the Pinus monticola Cr2 locus controlling resistance to white pine blister rust.

Molecular breeding incorporates efficient tools to increase rust resistance in five-needle pines. Susceptibility of native five-needle pines to white pine blister rust (WPBR), caused by the non-native invasive fungus Cronartium ribicola (J.C. Fisch.), has significantly reduced wild populations of these conifers in North America. Major resistance (R) genes against specific avirulent pathotypes have been found in several five-needle pine species. In this study, we screened genic SNP markers by comparative transcriptome and genetic association analyses and constructed saturated linkage maps for the western white pine (Pinus monticola) R locus (Cr2). Phenotypic segregation was measured by a hypersensitive reaction (HR)-like response on the needles and disease symptoms of cankered stems post inoculation by the C. ribicola avcr2 race. SNP genotypes were determined by HRM- and TaqMan-based SNP genotyping. Saturated maps of the Cr2-linkage group (LG) were constructed in three seed families using a total of 34 SNP markers within 21 unique genes. Cr2 was consistently flanked by contig_2142 (encoding a ruvb-like protein) and contig_3772 (encoding a delta-fatty acid desaturase) across the three seed families. Cr2 was anchored to the Pinus consensus LG-1, which differs from LGs where other R loci of Pinus species were mapped. GO annotation identified a set of NBS-LRR and other resistance-related genes as R candidates in the Cr2 region. Association of one nonsynonymous SNP locus of an NBS-LRR gene with Cr2-mediated phenotypes provides a valuable tool for marker-assisted selection (MAS), which will shorten the breeding cycle of resistance screening and aid in the restoration of WPBR-disturbed forest ecosystems.